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NIR-II hyperspectral imaging is a method for deep-tissue, high-resolution optical imaging in biological samples. The use of in vivo optical imaging with NIR-II probes is made feasible using upconverting nanoparticles (UCNPs) as fluorescent probes that emit in this range. UCNPs are lanthanide-doped fluoride nanoparticles that are better suited than conventional fluorophores for biomedical imaging purposes. Whereas the prior maximum reported depth of penetration of UCNPs was 3.2 cm, combining these probes with hyperspectral imaging allows for depths of up to ~9 cm in biological tissues. Hyperspectral imaging collects electromagnetic spectral information for each pixel in a 2D image. By obtaining the spatial coordinates and the wavelength of each pixel, the system can be used to generate 3D anatomical information from 2D scans. It can image at a range of scales from cells (tens of microns) to tumor microenvironments, organs, and the whole body. The system is a promising alternative to existing methods of clinical imaging modalities that suffer from limited ranges of detection and sensitivity.